PURINE METABOLISM IN M A N - I I I Biochemical, Immunological, and Cancer Research
E d i t e d by
Aurelio Rapado Fundacion Jimenez Diaz Madrid, Spain
R.W.E. Watts M.R.C. Clinical Research Centre Harrow, England
a n d
Chris H.M.M. De Bruyn Department of Human Genetics University of Nijmegen Faculty o f Medicine Nijmegen, The Netherlands
PLENUM PRESS · NEW YORK AND LONDON
Contents of Part Β
I . PURINE METABOLISM PATHWAYS AND REGULATION
A. De Novo S y n t h e s i s ; P r e c u r s o r s and R e g u l a t i o n
De Novo P u r i n e S y n t h e s i s i n C u l t u r e d Human F i b r o b l a s t s 1
R.B. Gordon, L. Thompson, L.A. Johnson, and B.T. Emmerson
Comparative M e t a b o l i s m o f a New A n t i l e i s h m a n i a l A g e n t , A l l o p u r i n o l R i b o s i d e , i n t h e P a r a s i t e and t h e Host C e l l 7
D. J. N e l s o n , S.W. LaFon, G.B. E l i o n , J . J . Ma r r , and R.L. Berens
P u r i n e M e t a b o l i s m i n Rat S k e l e t a l Muscle 13 E. R. T u l l y and T.G. Sheehan
A l t e r a t i o n s i n P u r i n e M e t a b o l i s m i n C u l t u r e d F i b r o b l a s t s w i t h HGPRT D e f i c i e n c y and w i t h PRPPP S y n t h e t a s e S u p e r a c t i v i t y 19
E. Z o r e f - S h a n i and 0. S p e r l i n g
P u r i n e M e t a b o l i s m i n C u l t u r e d E n d o t h e l i a l C e l l s 25 S. Nees, A.L. Gerbes, B. Willershausen-Zönnchen, and E. G e r l a c h
D e t e r m i n a n t s o f 5 - P h o s p h o r i b o s y l - l - P y r o p h o s p h a t e (PRPP) S y n t h e s i s i n Human F i b r o b l a s t s 31
K.0, R a i v i o , Ch. L a z a r , H. Krumholz, and M.A. Becker
X a n t h i n e O x i d o r e d u c t a s e I n h i b i t i o n by NADH as a R e g u l a t o r y F a c t o r o f P u r i n e M e t a b o l i s m 35
M.M. Jezewska and Z.W. K a m i n s k i
vii
viii C O N T E N T S OF P A R T Β
Β. N u c l e o t i d e M e t a b o l i s m
Human P l a c e n t a l Adenosine K i n a s e : P u r i f i c a t i o n and C h a r a c t e r i z a t i o n 41
CM. Andres, T.D. P a l e l l a , and I.H. Fox
Long-Term E f f e c t s o f Ribose on Adenine N u c l e o t i d e M e t a b o l i s m i n I s o p r o t e r e n o l - S t i m u l a t e d H e a r t s . . 45
H.-G. Zimmer, Η. I b e l , G. S t e i n k o p f f , and H. Kosc h i n e
C. Salvage Pathways
P u r i n e Salvage Enzymes i n Man and L e i s h m a n i a d o n o v a n i . . . 51 T.A. K r e n i t s k y , G.W. K o s z a l k a , J.V. T u t t l e , D. L. Adamczyk, G.B. E l i o n , and J . J . Marr
R e g u l a t i o n o f P u r i n e Salvage Enzymes i n Έ. c o l i 57
R.A. L e v i n e and M.W. T a y l o r
D. C a t a b o l i s m
P u r i n e T r a n s p o r t and t h e C e l l C y c l e 61 M.P. R i v e r a , M.R. Grau, J. R i g a u , and A. Goday
H y p o x a n t h i n e T r a n s p o r t i n Human E r y t h r o c y t e s 69 C. S a l e r n o and G i a c o m e l l o
Uptake o f Adenosine i n Human E r y t h r o c y t e s 73 M. Kraupp, P. Chiba, and M.M. Müller
E f f e c t o f A c t i n o m y c i n D on i n v i v o P u r i n e B i o s y n t h e s i s i n Hamster C e l l s 79
M.W. T a y l o r , K.C. Gupta, and L. Z a w i s t o w i c h
P u r i n e C a t a b o l i s m i n I s o l a t e d H e p a t o c y t e s : I n f l u e n c e o f C o f o r m y c i n 85
G. Van den Berghe, F. Bontemps, and H.G. Hers
I I . ENZYMOLOGY AND PURINE METABOLISM
A. P h o s p h o r i b o s y l t r a n s f e r a s e s
I n a c t i v a t i o n o f H y p o s a n t h i n e Guanine Phospho-r i b o s y l t r a n s f e r a s e by Guanosine D i a l d e h y d e : An A c t i v e S i t e D i r e c t e d I n h i b i t o r 87
L.A, Johnson, R.B, Gordon, and B.T. Emmerson
C O N T E N T S O F P A R T Β ix
Role o f Human H y p o x a n t h i n e Guanine P h o s p h o r i b o s y l -t r a n s f e r a s e i n N u c l e o t i d e I n t e r c o n v e r s i o n . . . . 93
A. G i a c o m e l l o and C. S a l e r n o
P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f Mammalian Adenine P h o s p h o r i b o s y l t r a n s f e r a s e 103
M.W. T a y l o r and H.V. Hershey
The E f f e c t o f P h o s p h o r i b o s y l p y r o p h o s p h a t e on S t a b i l i t y and C o n f i g u r a t i o n o f Hypoxan-t h i n e g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e and A d e n i n e p h o s p h o r i b o s y l t r a n s f e r a s e f r o m Human E r y t h r o c y t e s I l l
W. Gröbner and N. Zöllner
C h e m i c a l M o d i f i c a t i o n o f Hypoxanthine-phospho-r i b o s y l t r a n s f e r a s e and I t s P r o t e c t i o n by S u b s t r a t e s and P r o d u c t s 117
W.. Gutensohn and H. Jahn
Β.. N u c l e o s i d e P h o s p h o r i b o s y l a t i n g Enzymes
S t r u c t u r a l S t u d i e s o f Human Adenine P h o s p h o r i b o s y l -t r a n s f e r a s e P u r i f i e d by A f f i n i t y Chromatography 123
J,A. H olden, G.S. M e r e d i t h , and W.N. K e l l e y
P h o s p h o r i b o s y l p y r o p h o s p h a t e (PRPP) S y n t h e t a s e M utant i n S a l m o n e l l a t y p h i m u r i u m 131
B,. Jochimsen, Β. Garber, and J.S. Gots
C. N u c l e o s i d e C l e a v i n g Enzymes
M e t h y l m e r e a p t o p u r i n e R i b o n u c l e o s i d e T o x i c i t y i n Human F i b r o b l a s t s : I n h i b i t i o n o f Phosphor i b o s y l p y r o p h o s p h a t e S y n t h e t a s e as w e l l as A m i d o p h o s p h o r i b o s y l t r a n s f e r a s e 137
R..C.K. Yen and M.A. Becker
A d e n o s i n e K i n a s e : R e g u l a t i o n by S u b s t r a t e s , Magnesium, and pH 145
R..L. M i l l e r and D.L. Adamczyk
A d e n o s i n e and Deoxyadenosine K i n a s e f r o m Rat L i v e r . . . . 151 N.. Ogasawara, Y. Yamada, and H. Goto
D- D e a m i n a t i n g Enzymes
R a d i o immunochemical A n a l y s i s o f Human E r y t h r o c y t e Adenosine Deaminase
P..E. Daddona, M.A. Frohman, and W.N. K e l l e y 157
χ C O N T E N T S O F P A R T Β
Adenosine Deaminase C o n v e r s i o n P r o t e i n s : A P o t e n t i a l Role 163
P.P. T r o t t a and M.E. B a l i s
R e g u l a t o r y P r o p e r t i e s o f AMP Deaminase Isozymes 169 N. Ogasawara, H. Goto, and Y. Yamada
Human Adenosine Deaminase: S t o i c h i o m e t r y o f t h e Large Form Complex 177
P.E. Daddona and W.N. K e l l e y
Guanase f r o m Human L i v e r - P u r i f i c a t i o n and C h a r a c t e r i z a t i o n 183
R. K u z m i t s , H. Stemberger, and M.M. Müller
Ε. O x i d a t i n g Enzymes
N i c o t i n a m i d e and L i v e r X a n t h i n e Oxidase 189 A. D i S t e f a n o , Μ. P i z z i c h i n i , and Ε. M a r i n e l l o
X a n t h i n e Oxidase A c t i v i t y i n Human I n t e s t i n e s . H i s t o c h e m i c a l and R a d i o c h e m i c a l Study 197
C. Auseher, Ν. Amory, P. van der Kemp, and F. D e l b a r r e
I I I . PYRIMIDINE METABOLISM
P u r i n e and P y r i m i d i n e M e t a b o l i s m i n H e r e d i t a r y O r o t i c a c i d u r i a D u r i n g a 15-Year Follow-Up Study 203
D. R. Webster, H.A. Simmonds, C.F. P o t t e r , and D.M.O. B e c r o f t
E f f e c t o f A l l o p u r i n o l on P y r i m i d i n e M e t a b o l i s m i n Human Whi t e B l o o d C e l l s : Role o f t h e Salvage Pathway 209
P. B a n h o l z e r , W. Gröbner, and N. Zöllner
K i n e t i c s and Co m p a r t m e n t a t i o n o f E r y t h r o c y t e P y r i m i d i n e M e t a b o l i s m 217
E. H. H a r l e y , P. Z e t l e r , and S. Ne a l
S i m u l t a n e o u s D e t e r m i n a t i o n o f Rates o f P u r i n e and P y r i m i d i n e S y n t h e s i s i n C u l t u r e d Human Lymphoblasts and F i b r o b l a s t s 223
W.H. Huisman, K.O. R a i v i o , and M.A. Becker
A c t i v i t i e s o f Enzymes o f P u r i n e and P y r i m i d i n e M e t a b o l i s m i n Nine Mycoplasma Species 231
M. Hamet, C. B o n i s s o l , and P. C a r t i e r
CONTENTS O F P A R T Β x«
I V . LYMPHOCYTE PURINE METABOLISM RESEARCH
I n c r e a s e s i n P u r i n e E x c r e t i o n and Rate o f S y n t h e s i s by Drugs I n h i b i t i n g IMP Dehydrogenase o r A d e n y l o s u c c i n a t e S y n t h e t a s e A c t i v i t i e s 237
R.C. W i l l i s and J.E. S e e g m i l l e r
P o s s i b l e Role f o r 5 T - N u c l e o t i d a s e i n Deoxyadenosine S e l e c t i v e T o x i c i t y t o C u l t u r e d Human Lymp h o b l a s t s 243
R.L. Wortmann, B.S. M i t c h e l l , N.L. Edwards, and I.H. Fox
C y c l i c N u c l e o t i d e L e v e l s and Mechanism o f I n h i b i t i o n o f L e u c o c y t e F u n c t i o n by Adenosine Deaminase I n h i b i t i o n 251
A.D. M e i s e l , Ch. N a t a r a j a n , G. S t e r b a , and H.S. Diamond
P u r i n e R i b o n u c l e o s i d e and D e o x y r i b o n u c l e o s i d e M e t a b o l i s m i n Thymocytes 259
F.F. Snyder and T. Lukey
M o l e c u l a r Mechanism(s) o f D e o x y r i b o n u c l e o s i d e i n T-Lymphoblasts 265
J.M. W i l s o n , B.S. M i t c h e l l , and W.N. K e l l e y
I n h i b i t i o n o f Immune C e l l F u n c t i o n by Adenosine: B i o c h e m i c a l S t u d i e s 271
T.P. Zimmerman, G. WoIberg, G.S. Duncan, R.D. Deeprose, and R.J. Harvey
I n t e r a c t i o n s Between Energy M e t a b o l i s m and Adenine N u c l e o t i d e M e t a b o l i s m i n Human Lymphoblasts 277
S.S. Matsumoto, K.O. R a i v i o , R.C. W i l l i s , and J.E. S e e g m i l l e r
Enzymes o f P u r i n e I n t e r c o n v e r s i o n s i n S u b f r a c t i o n s o f Lymphocytes 283
J.P.R.M. van La a r h o v e n , G.Th. S p i e r e n b u r g , C.H.M.M. De B r u y n , and E.D.A.M. S c h r e t l e n
Measurement o f t h e Rates o f S y n t h e s i s and D e g r a d a t i o n o f H y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l -t r a n s f e r a s e i n Human Lymphoblasts 289
P. Moore M a t t e s and W.N. K e l l e y
xü C O N T E N T S O F PART Β
Human 5 1 - N u c l e o t i d a s e . P r o p e r t i e s and C h a r a c t e r i z a t i o n o f t h e Enzyme f r o m P l a c e n t a . Lymphocytes and L y m p h o b l a s t o i d C e l l s i n C u l t u r e 295
W. Gutensohn
M e t a b o l i s m and T o x i c i t y o f 9-Beta-D-Arabino-f u r a n o s y l a d e n i n e i n Human M a l i g n a n t Τ C e l l s and Β C e l l s i n T i s s u e C u l t u r e 299
D. A. Carson, J. Kaye, and J.E. S e e g m i l l e r
V. IMMUNE SYSTEM AND PURINE METABOLISM
S u p p r e s s i o n o f C e l l u l a r I m m unity Due t o I n h i b i t i o n o f P u r i n e N u c l e o s i d e P h o s p h o r y l a s e by A l l o p u r i n o l - R i b o s i d e 309
Y. N i s h i d a , N. Kamatani, K. Tanimoto, and I . Akaoka
Lymphocyte 5 T - N u c l e o t i d a s e D e f i c i e n c y : C l i n i c a l and M e t a b o l i c C h a r a c t e r i s t i c s o f t h e A s s o c i a t e d Hypogammaglobulinemia 315
N.L. Edwards, J.T. Cas s i d y , and I.H. Fox
I m m u n o l o g i c a l S t u d i e s on Lesch-Nyhan P a t i e n t s 321 C.H.M.M. de Bruyn , Ph. Gausset, J. Duchateau, E. Vamos, S. K u l a k o w s k i and G. Delespesse
A c t i v i t y o f E c t o - 5 T - N u c l e o t i d a s e i n L y m p h o b l a s t o i d C e l l L i n e s D e r i v e d f r o m C a r r i e r s o f C o n g e n i t a l X-Linked Agammaglobulinemia 327
L.F. Thompson, G.R. Boss, A. B i a n c h i n o , and J.E. S e e g m i l l e r
V I . CANCER RESEARCH
Adenosine Deaminase and P u r i n e N u c l e o s i d e P h o s p h o r y l a s e i n Ac u t e and C h r o n i c L y m p h a t i c Leukemia 333
H. Lud w i g , Η. W i n t e r l e i t n e r , R. K u z m i t s , and M.M. Müller
P u r i n e Salvage Enzymes i n Lymphocytes and G r a n u l o c y t e s f r o m P a t i e n t s w i t h S m a l l - C e l l Carcinoma o f t h e Lung 339
P. Nygaard and J. M e j e r
CONTENTS OF P A R T Β xiii
Treatment o f Acut e L y m p h o b l a s t i c Leukemia w i t h t h e Adenosine Deaminase I n h i b i t o r 2 r - D e o x y c o f o r m y c i n 347
B.S. M i t c h e l l , Ch.A. K o l l e r , and W.N. K e l l e y
I n c r e a s e o f P h o s p h o r i b o s y l p y r o p h o s p h a t e L e v e l s i n C u l t u r e d L1210 Leukemia C e l l s Exposed t o M e t h o t r e x a t e 351
J.M. Buesa, A. Leyva, and H.M. Pinedo
P u r i n e Salvage Pathway i n Leukemic C e l l s 357 A. Goday, M.R. Grau, I . J a d r a q u e , and M.P. R i v e r a
B i o c h e m i c a l Consequences o f Tre a t m e n t w i t h t h e Adenosine Deaminase I n h i b i t o r 2 ' - Deoxycoformycin 365
R.M. P a i n e , J.F. Smyth, and K.R. Harrap
I n v i t r o and i n v i v o E f f e c t o f D e o x y c o f o r m y c i n i n Human Τ C e l l Leukemia 373
A.L. Yu, F.H. Kung, B. Bakay, and W.L. Nyhan
Uniqueness o f D e o x y r i b o n u c l e o t i d e M e t a b o l i s m i n Human M a l i g n a n t Τ C e l l L i n e s 381
D.A. Carson, J . Kaye, S. Matsumoto, J.E. S e e g m i l l e r , and L. Thompson
V I I . METHODOLOGY
Hi g h Performance L i q u i d Chromatography o f Plasma P y r i m i d i n e s and P u r i n e s and I t s A p p l i c a t i o n i n Cancer Chemotherapy 389
A. Leyva, J . S c h o r n a g e l , and H.M. Pinedo
U r a t e - B i n d i n g P r o t e i n s i n Plasma S t u d i e d by A f f i n i t y Chromatography 395
M.L. Ciompi, A. L u c a c c h i n i , D. S e g n i n i , and M.R. Mazzoni
Chromatographic D e t e r m i n a t i o n o f PRPP-Synthetase A c t i v i t y i n Human B l o o d C e l l s 401
P. Nygaard and K.F. Jensen
P u r i f i c a t i o n o f M y o c a r d i a l Adenosine K i n a s e Using A f f i n i t y and Ion-Exchange Chromatography . . - . 409
M.P. U i t e n d a a l , J.W. De Jong, E. Harmsen, and E. K e i j z e r
xiv C O N T E N T S O F P A R T Β
M i c r o m e t h o d s f o r t h e Measurement o f P u r i n e Enzymes i n Lymphocytes 415
J.P.R.M. v a n L a a r h o v e n , G.Th. S p i e r e n b u r g , F.T.J.J. Oerlemans, and C.H.M.M. De Bruyn
A R a p i d S c r e e n i n g Method f o r I n b o r n E r r o r s o f P u r i n e and P y r i m i d i n e M e t a b o l i s m U s i n g I s o t a c h o p h o r e s i s 421
H.A. Simmonds, A. Sahota, and R. Payne
A n a l y s i s o f Serum P u r i n e s and P y r i m i d i n e s by I s o t a c h o p h o r e s i s 429
F. Oerlemans, Th. Verheggen, F. M i k k e r s , F. E v e r a e r t s , and C.H.M.M. De Bruyn
D e t e r m i n a t i o n o f U r i c A c i d i n Serum: Comparison o f a St a n d a r d Enzymatic Method and I s o t a c h o p h o r e s i s 435
F. Oerlemans, Th. Verheggen, F. M i k k e r s , F. E v e r a e r t s , and C.H.M.M. De Bruyn
A d d i t i o n P r o d u c t s o f U r i c A c i d and Formaldehyde 441 P.A. S i m k i n and Q.P. Lee
Automated R e t r i e v a l o f P u r i n e L i t e r a t u r e 445 L. F e r r e i r o and A. Rey
A u t h o r I n d e x 449
S u b j e c t I n d e x 453
Contents of Part A
I . CLINICAL GOUT
The N a t u r a l H i s t o r y o f H y p e r u r i c e m i a Among A s y m p t o t i c R e l a t i v e s o f P a t i e n t s W i t h Gout . 1
T.-F. Yii and C. Kaung
The C l i n i c a l D i f f e r e n t i a t i o n o f P r i m a r y Gout From P r i m a r y Renal Disease i n P a t i e n t s W i t h B o t h Gout and Renal Disease 9
Β. T. Emmerson, P. J. S t r i d e and G. W i l l i a m s
Renal F a i l u r e i n Young S u b j e c t s W i t h F a m i l i a l Gout 15
H. A. Simmonds, J . S. Cameron, C. F. P o t t e r , D. Warren, T. G i b s o n , and D. F a r e b r o t h e r
F a m i l y Study o f L i p i d and P u r i n e L e v e l s i n Gout P a t i e n t s and A n a l y s i s o f M o r t a l i t y 21
L. G. D a r l i n g t o n , J. S l a c k , and J. T. S c o t t
U r i c A c i d T u r n o v e r i n Normals, i n Gout and i n C h r o n i c Renal F a i l u r e U s i n g 1 4 C - U r i c A c i d 27
C. V i t a l i , G. Pasero, A, C l e r i c o , L. R i e n t e , N. Molea, A. P i l o , G. M a r i a n i , and R. B i a n c h i
E r y t h r o c y t e Adenosine-Deaminase A c t i v i t y i n Gout and H y p e r u r i c e m i a 33
A. C a r c a s s i , P. M a c r i , G. C h i a r o n i , and S. B o s c h i
C l i n i c a l V a r i a b i l i t y o f t h e Gouty D i a t h e s i s 39 W. J. A r n o l d and R. A. Simmons
xv
xvi CONTENTS O F P A R T A
C l i n i c a l F e a t u r e s o f 4,000 Gouty S u b j e c t s i n Japan 47
N. N i s h i o k a and K. M i k a n a g i
Frequency o f C h o n d r o c a l c i n o s i s o f t h e Knees and A v a s c u l a r N e c r o s i s o f t h e Femoral Heads i n Gout, a C o n t r o l l e d Study 55
A. Stockman, L. G. D a r l i n g t o n , and J. T. S c o t t
Gaschromatographic E v a l u a t i o n o f U r i n a r y 1 7 - K e t o s t e r o i d s , E t i o c h o l a n o l o n e and D e h y d r o e p i a n d r o s t e r o n e i n P r i m a r y Gout and H y p e r u r i c e m i a 59
A. C a r c a s s i , F. L o r e , G. Manasse, P. M a c r i , and M. Pisano
Hormonal A s p e c t s o f Gouty P a t i e n t s 65 U. V a l e n t i n i , G. R i a r i o - S f o r z a , R. Ma r c o l o n g o , and E. M a r i n e l l o
D e t e r m i n a t i o n o f T u b u l a r S e c r e t i o n o f U r a t e i n H e a l t h y and Gouty Men 73
L. B. Sorensen and D. J. L e v i n s o n
Ribose T o l e r a n c e i n Gouty P a t i e n t s 81 M. P i z z i c h i n i , R. M a r c o l o n g o , and E. M a r i n e l l o
I I . URIC ACID AND RENAL STONES
U r i n a r y U r a t e and U r i c A c i d R e l a t i v e S a t u r a t i o n i n N o r m o u r i c u r i c C a l c i u m O x a l a t e Stone Formers W i t h Normal U r i n a r y C a l c i u m O x a l a t e S a t u r a t i o n 87
M. Labeeuw, C. G e r b a u l e t , N. P o z e t , P. Zech, and J. T r a e g e r
C o r r e l a t i o n Between t h e U r i c A c i d and C a l c i u m C o n c e n t r a t i o n i n U r i n e . R e s u l t s o f a Long Term Study on R e c u r r e n t Stone-Formers and H e a l t h y C o n t r o l s 93
P. L e s k o v a r , R. Härtung, and M. K r a t z e r
I n t e r a c t i o n o f H y p e r u r i c u r i a and H y p e r o x a l u r i a on R e nal C a l c i u m O x a l a t e Stone F o r m a t i o n 99
F. H e r i n g , Κ.-Η. B i g a l k e , and W. L u t z e y e r
U r i c A c i d / C a l c i u m O x a l a t e N e p h r o l i t h i a s i s . C l i n i c a l and B i o c h e m i c a l F i n d i n g s i n 86 P a t i e n t s 109
A. Rapado, J.M. C a s t r i l l o , M. D i a z - C u r i e l , M. L. Traba, M. Santos, L. C i f u e n t e s - D e l a t t e
C O N T E N T S OF P A R T Α ^ , .
The U r i c A c i d : C y s t i n e C o r r e l a t i o n i n t h e U r i n e o f R e c u r r e n t C a l c i u m O x a l a t e Stone-Formers and H e a l t h y C o n t r o l s 115
P. L e s k o v a r , R. Härtung, and M. K r a t z e r
The Role o f U r a t e i n I d i o p a t h i c C a l c i u m U r o l i t h i a s i s 121
S. R. S i l c o c k
M i n e r a l o g i e C o m p o s i t i o n o f 66 Mixed U r i n a r y C a l c u l i o f C a l c i u m O x a l a t e and U r i c A c i d 129
J. R. M i n o n - C i f u e n t e s , M. Santos, and L. C i f u e n t e s - D e l a t t e
H y p e r u r i c e m i a and C y s t i n u r i a 135 F. L i n a r i , M. M a r a n g e l l a , B. M a l f i , G. Vacha, Μ. Bruno, G. G i o r c e l l i , and B. F r u t t e r o
Monosodium U r a t e Monohydrate as S p h e r u l i t e s 141 J . J. F i e c h t n e r and P. A. S i m k i n
I I I . CLINICAL AND PHYSIOLOGICAL ASPECTS OF PURINE METABOLISM
Tumoural H y p o u r i c e m i a 145 A. Lesmes, M. D i a z - C u r i e l , and J. M. C a s t r i l l o
H e r e d i t a r y Renal H y p o u r i c e m i a W i t h H y p e r u r i c o s u r i a and V a r i a b l y A b s o r p t i v e H y p e r c a l c i u r i a and U r o l i t h i a s i s - A New Syndrome 149
0. S p e r l i n g and A. de V r i e s
H e r e d i t a r y and E n v i r o n m e n t a l F a c t o r s I n f l u e n c i n g on t h e Serum U r i c A c i d Throughout Ten Years P o p u l a t i o n Study i n Japan 155
K. N i s h i o k a and K. M i k a n a g i
The N a t u r a l H i s t o r y o f U r a t e O v e r p r o d u c t i o n i n S i c k l e C e l l Anemia 161
H. S. Diamond, A. D. M e i s e l , and D. Holden
Salvage Pathway i n E r y t h r o c y t e s o f P a t i e n t s W i t h P s o r i a s i s 167
G. P a r t s c h , F. Mayer, R. E b e r l , and A. Luger
Serum 5 - N u c l e o t i d a s e i n P r o g r e s s i v e M u s c u l a r D y s t r o p h y 173
F. Lähoda and K. B a i e r
xviü C O N T E N T S O F P A R T A
P u r i n e M e t a b o l i s m i n Duchenne M u s c u l a r D y s t r o p h y 177
C. Η. Μ. M. De B r u y n , S. K u l a k o w s k i , C. A. van Bennekom, P. R e n o i r t e , and Μ. M. Müller
M e t a b o l i s m o f Adenine and Adenosine i n E r y t h r o c y t e s o f P a t i e n t s w i t h M y o t o n i c M u s c u l a r D y s t r o p h y (MMD) 183
Μ. M. Müller, Μ. F r a s s , and B. Mamoli
C l i n i c a l and E n z y m o l o g i c a l S t u d i e s i n a C h i l d W i t h Type I Glycogen S t o r a g e Disease A s s o c i a t e d W i t h P a r t i a l D e f i c i e n c y o f H e p a t i c Glucose-6-Phosphatase 189
G. N u k i and J . P a r k e r
D i f f e r e n t i a l A b s o r p t i o n o f P u r i n e N u c l e o t i d e s , N u c l e o s i d e s and Bases 203
C. F. P o t t e r , A. Cadenhead, H. A. Simmonds, and J. S. Cameron
I n f l u e n c e o f D i e t a r y P r o t e i n on Serum and U r i n a r y U r i c A c i d 209
W. Löffler, W. Gröbner, and N. Zöllner
E f f e c t o f H y p o x a n t h i n e i n Meat on Serum U r i c A c i d and U r i n a r y U r i c A c i d E x c r e t i o n 215
W. K. Spann, W. Gröbner, and N. Zöllner
On t h e Mechanism o f t h e P a r a d o x i c a l E f f e c t o f S a l i c y l a t e on U r a t e E x c r e t i o n 221
H. S. Diamond, G. S t e r b a , K. Jayadeven, and A. D. M e i s e l
The U r i c o s u r i c A c t i o n o f P r o t e i n i n Man 227 F. M a t z k i e s , G. Berg, and H. Mädl
Hypoxic E f f e c t s on P u r i n e M e t a b o l i s m S t u d i e d W i t h H i g h P r e s s u r e L i q u i d Chromatography 233
R. A. Harkness, R. J. Simmonds, and M. C. O'Connor
U r a t e M e t a b o l i s m i n a M o n g r e l Dog 237 P. A. S i m k i n
COINTENTS O F P A R T A x i x
I V . STUDIES ON XANTHINURIA
Thie E f f e c t o f Weight R e d u c t i o n on Plasma and U r i n a r y L e v e l s o f O x y p u r i n e s i n an Obese X a n t h i n u r i c P a t i e n t 241
C. A u s c h e r , C. P a s q u i e r , N. Amory, G. Gay, A. A i s e n e , and G. Debry
X a m t h i n u r i a : The Cause o f H y p o u r i c e m i a i n H e p a t i c Disease 247
H. J. Castro-Mendoza, A. Rapado, C. De La P i e d r a , and J. M. C a s t r i l l o
X a i n t h i n e - C o p r o p o r p h y r i n I I I 251 H. J. Castro-Mendoza
V. THERAPEUTICAL ASPECTS OF DISORDERS IN PURINE METABOLISM
A C o n t r o l l e d Study o f t h e E f f e c t o f Long Term A l l o p u r i n o l T r e a t m e n t on Renal F u n c t i o n i n Gout 257
T. G i b s o n , H. A. Simmonds, C. P o t t e r , and V. Rogers
St:udies W i t h A l l o p u r i n o l i n P a t i e n t s W i t h I m p a i r e d Renal F u n c t i o n 263
G. B. E l i o n , F. M. Benezra, T. D. Beardmore, and W. N. K e l l e y
P h i a r m a c o l o g i c a l E f f e c t s o f 1 , 3 , 5 - T r i a z i n e s and T h e i r E x c r e t i o n C h a r a c t e r i s t i c s i n t h e Rat 269
M. H r o p o t , F. Sörgel, Β. v. Kerekjärto, H. J. Lang, and R. Muschaweck
T i i e n i l i c A c i d i n t h e T r e a t m e n t o f Gout and H y p e r t e n s i o n 277
T. Gibson, C. P o t t e r , H. A. Simmonds, V. Rogers, and R. I . G l e a d l e
Benzbromarone as a Long-Term U r i c o s u r i c Agent 283 R. B l u e s t o n e , J. K l i n e n b e r g , and I . K. Lee
Thie A c t i o n o f Benzbromarone i n R e l a t i o n t o Age, Sex and Accompanying Diseases 287
H. F e r b e r , U. Bader, and F. M a t z k i e s
Covalitin®: A New Drug f o r t h e T r e a t m e n t o f U r i c L i t h i a s i s 295
T. C o v a l i u
XX C O N T E N T S O F P A R T A
V I . MUTATIONS AFFECTING PURINE METABOLISM
A. P h o s p h o r i b o s y l t r a n s f e r a s e s
H y p o x a n t h i n e Salvage i n Man: I t s I m p o r t a n c e i n U r a t e O v e r p r o d u c t i o n i n t h e Lesch-Nyhan Syndrome 301
N. L. Edwards, D. P. Recker, and I . H. Fox
As p e c t o f P u r i n e M e t a b o l i c A b e r r a t i o n A s s o c i a t e d W i t h U r i c A c i d O v e r p r o d u c t i o n and Gout 307
L. C. Y i p , T.-F. Yü, and Μ. E. B a l i s
P r o p e r t i e s o f a Mutant H y p o x a n t h i n e - P h o s p h o r i b o s y l -t r a n s f e r a s e i n a P a t i e n t W i t h Gout 313
W. Gröbner and W. Gutensohn
V a r i a t i o n i n Human HPRT and I t s R e l a t i o n s h i p t o N e u r o l o g i c and B e h a v i o r a l M a n i f e s t a t i o n s 317
B. Bakay, E. N i s s i n e n , L. Sweetman, U. Franeke, and W. L. Nyhan
H i g h HPRT A c t i v i t y i n F i b r o b l a s t s f r o m P a t i e n t s W i t h Lesch-Nyhan Syndrome due t o B a c t e r i a l "L-Form" C o n t a m i n a t i o n 327
I . W i l l e r s , S, S i n g h , K. R. H e l d , and H. W. Goedde
K i n e t i c s o f a HGPRT Mu t a n t Showing S u b s t r a t e I n h i b i t i o n 333
Ε. H. H a r l e y , C. M. Adnams, and L. M. Steyn
B. N u c l e o s i d e P h o s p h o r i b o s y l a t i n g Enzymes
Spectrum o f 2,8 - D i h y d r o x y a d e n i n e U r o l i t h i a s i s i n Complete APRT D e f i c i e n c y 337
H. A. Simmonds, T. M. B a r r a t t , D. R. Webster, A. Sahota, K. J. Van A c k e r , J. S. Cameron, and M. D i l l o n
Complete Adenine P h o s p h o r i b o s y l t r a n s f e r a s e (APRT) D e f i c i e n c y i n Two S i b l i n g s : Report o f a New Case 343
P. C a r t i e r , M. Hamet, A. V i n c e n s , J. L. P e r i g n o n
I n h e r i t a n c e o f Adenine P h o s p h o r i b o s y l t r a n s f e r a s e (APRT) D e f i c i e n c y 349
K. J. Van A c k e r , H. A. Simmonds, C. F. P o t t e r , and A. Sahota
CCHNTENTS OF P A R T A xxi
I i m m u n o l o g i c a l E v a l u a t i o n o f a F a m i l y D e f i c i e n t i n A d e n i n e P h o s p h o r i b o s y l T r a n s f e r a s e (APRT) 355
W. J. S t e v e n s , Μ. E. Peetermans, and K. J. Van Acker
A c t i v i t i e s o f A m i d o p h o s p h o r i b o s y l t r a n s f e r a s e and P u r i n e P h o s p h o r i b o s y l t r a n s f e r a s e s i n D e v e l o p i n g Rat B r a i n 361
J. A l l s o p and R. W. E. Watts
P u i r i n e N u c l e o s i d e P h o s p h o r y l a s e D e f i c i e n c y ; G e n e t i c S t u d i e s i n a D u t c h F a m i l y 367
G. E. J. S t a a l , M. J. M. van der V l i s t , R. G e e r d i n k , J. M. J a n s e n - S c h i l l h o r n van Veen, B. J. M. Z e g e r s , and J. W. Stoop
C. N u c l e o s i d e C l e a v i n g Enzymes
A b n o r m a l R e g u l a t i o n o f P u r i n e M e t a b o l i s m i n a C u l t u r e d Mouse T - C e l l Lymphoma Mut a n t P a r t i a l l y D e f i c i e n t i n A d e n y l o s u c c i n a t e S y n t h e t a s e 375
B. U l l m a n , M. A. Wormsted, B. B. L e v i n s o n , L. J. Gudas, A. Cohen, S. M. C l i f t , and D. W. M a r t i n , J r .
S u i p e r a c t i v e P h o s p h o r i b o s y l p y r o p h o s p h a t e S y n t h e t a s e W i t h A l t e r e d R e g u l a t o r y and C a t a l y t i c P r o p e r t i e s 387
M. A. B ecker, K. 0. R a i v i o , B. Bakay, W. B. Adams, and W. L. Nyhan
AM1P Phosphatase A c t i v i t y i n Human Term P l a c e n t a : S t u d i e s on P l a c e n t a l 5 f - N u c l e o t i d a s e 393
Μ. H. M a g u i r e and T. P. K r i s h n a k a n t h a
D. D e a m i n a t i n g Enzymes
Adlenosine and Deoxyadenosine M e t a b o l i s m i n t h e E r y t h r o c y t e s o f a P a t i e n t W i t h Adenosine Deaminase D e f i c i e n c y 397
A. Sahota, H. A. Simmonds, C. F. P o t t e r , J . G. Watson, K. Hugh-Jones, and D. P e r r e t t
Complete Adenosine Deaminase (ADA) D e f i c i e n c y W i t h o u t I m m u n o d e f i c i e n c y , and P r i m a r y H y p e r o x a l u r i a , i n a 12-Year-01d Boy 403
J. L. P e r i g n o n , M. Hamet, P. C a r t i e r , and C. G r i s c e l l i
xxii CONTENTS OF PART A
M e t a b o l i s m o f Adenosine and Deoxyadenosine by S t o r e d Human Red C e l l s 409
G. R. B a r t l e t t
Adenosine Deaminase and P u r i n e N u c l e o s i d e P h o s p h o r y l a s e A c t i v i t i e s D u r i n g C u l t u r i n g o f F i b r o b l a s t s 415
M. P. U i t e n d a a l , F. T. J. J. Oerlemans, C. Η. Μ. M. De B r u y n , T . L. O e i , and P. Hösli
S-Adenosylhomocysteine M e t a b o l i s m i n Adenosine Deaminase D e f i c i e n t C e l l s 421
M. S. H e r s h f i e l d and Ν. M. K r e d i c h
A l t e r e d D e o x y n u c l e o s i d e T r i p h o s p h a t e L e v e l s P a r a l l e l i n g Deoxyadenosine T o x i c i t y i n Adenosine Deaminase I n h i b i t e d Human Lymphocytes 427
H. G. B l u e s t e i n , L. F. Thompson, D. A. A l b e r t , and J. E. S e e g m i l l e r
A u t h o r I n d e x 433
S u b j e c t I n d e x 437
PURINE METABOLISM I N CULTURED CORONARY ENDOTHELIAL CELLS
S. Nees, A.L. Gerbes, B. Willershausen-Zönnchen and E. G e r l a c h
P h y s i o l o g i s c h e s I n s t i t u t d er Universität München
P e t t e n k o f e r s t r . 12, D-8000 München 2
A l t h o u g h i t i s w e l l known t h a t e n d o t h e l i a l c e l l s a r e i n v o l v e d i n s e v e r a l b i o l o g i c a l p r o c e s s e s such as t r a n s p o r t * ! , h e m o s t a s i s ^ , s y n t h e s i s o f c o l l a g e n - ^ , h i s t a m i n e ^ and p r o s t a g l a n d i n s ^ , our knowledge c o n c e r n i n g i n t e r m e d i a r y m e t a b o l i s m o f t h e e n d o t h e l i u m i s r a t h e r l i m i t e d . I n t h e co u r s e o f s t u d i e s on i n t e r r e l a t i o n s h i p s b e t ween h e a r t f u n c t i o n and c a r d i a c m e t a b o l i s m ^ 7 w e became i n t e r e s t e d i n some f e a t u r e s o f p u r i n e m e t a b o l i s m o f c o r o n a r y e n d o t h e l i a l c e l l s . Our i n t e r e s t was i n i t i a t e d by t h e a s s u m p t i o n t h a t these c e l l s m i g h t c o n t r i b u t e t o t h e p r o d u c t i o n o f v a s o a c t i v e adenosine w h i c h i s c o n s i d e r e d t o p l a y an i m p o r t a n t r o l e i n t h e m e t a b o l i c r e g u l a t i o n o f c o r o n a r y b l o o d flow^»^. The s t u d i e s - n o t p o s s i b l e o f c o u r s e t o be p e r f o r m e d under i n v i v o c o n d i t i o n s - were c a r r i e d o u t on c u l t u r e d e n d o t h e l i a l c e l l s i s o l a t e d f r o m c o r o n a r y v e s s e l s o f g u i n e a p i g h e a r t s as r e c e n t l y d e s c r i b e d ^ .
MATERIALS AND METHODS
C u l t u r e Medium 199 (Seromed, München) c o n t a i n i n g p e n i c i l l i n e (200 TJ/ml) and s t r e p t o m y c i n e (200 ug/ml) was supplemented w i t h f e t a l c a l f serum ( 2 0 % ) and L - g l u t a m i n e (2 mM). Column p a c k i n g s ( t o t a l l y p o r o u s s i l i c a ) f o r H i g h P r e s s u r e L i q u i d Chromatography (HPLC) were o b t a i n e d f r o m Macherey & N a g e l , Düren. N u c l e o t i d e s , n u c l e o s i d e s and bases f o r c a l i b r a t i o n were purc h a s e d f r o m B o e h r i n g e r Mannheim, a l l o t h e r m a t e r i a l s o f h i g h e s t a v a i l a b l e p u r i t y f r o m Merck, D a r m s t a d t .
P r e p a r a t i o n o f c e l l s and c e l l c u l t u r e : Guinea p i g h e a r t s were c a n n u l a t e d t h r o u g h t h e a o r t a , and t h e i r c o r o n a r y system, washed
25
26 S. N E E S E T A L .
f r e e of b l o o d , was f i l l e d w i t h an i s o t o n i c b u f f e r s o l u t i o n c o n t a i n i n g c o l l a g e n a s e and t r y p s i n ( 0 . 1 % e a c h ) . A f t e r an exp o s u r e of 20 min p e r f u s i o n was s t a r t e d a g a i n and a l l e n d o t h e l i a l c e l l s w h i c h had been d e t a c h e d , were c o l l e c t e d f r o m t h e p e r f u s a t e by c e n t r i f u -g a t i o n . S u b s e q u e n t l y , t h e c e l l s were washed w i t h c u l t u r e medium and seeded i n c u l t u r e d i s h e s . C u l t i v a t i o n was p e r f o r m e d a t 3 7°C i n a h u m i d i f i e d a i r atmosphere c o n t a i n i n g 3% C O 2 . Depending on t h e i n o c u l u m c o n f l u e n c y was reached a f t e r 2 t o 4 weeks. C o n t a m i n a t i o n s w i t h f i b r o b l a s t s and smooth muscle c e l l s were u s u a l l y l e s s t h a n 2% As j u d g e d by e l e c t r o n m i c r o s c o p y t h e c u l t i v a t e d e n d o t h e l i a l c e l l s r e v e a l e d i m p o r t a n t m o r p h o l o g i c a l c r i t e r i a o f e n d o t h e l i a l c e l l s i n v i v o ( c l u s t e r s o f f r e e r i b o s o m e s , smooth and roug h e n d o p l a s m i c r e t i c u l u m , clumps o f co a r s e f i l a m e n t s , f i n e f i l a m e n t s and p r o m i n e n t m i c r o t u b u l e s i n t h e c y t o p l a s m a ) .
A n a l y s i s o f n u c l e o t i d e s , n u c l e o s i d e s and bases: C u l t u r e d end o t h e l i a l c e l l s were e x t r a c t e d w i t h 0.4 Ν p e r c h l o r i c a c i d . Q u a n t i t a t i o n o f t h e d i f f e r e n t p u r i n e compounds i n t h e n e u t r a l i z e d c e l l e x t r a c t s was c a r r i e d o u t by a p p l i c a t i o n o f s p e c i a l l y e l a b o r a t e d HPLC-techniques u s i n g weak a n i o n exchange columns f o r t h e s e p a r a t i o n o f t h e n u c l e o t i d e s and r e v e r s e phase columns f o r n u c l e o s i d e s and bases.
D e t e r m i n a t i o n o f enzyme a c t i v i t i e s : S p e c i f i c a c t i v i t i e s o f enzymes i n v o l v e d i n n u c l e o t i d e m e t a b o l i s m were measured i n a 20 000 g membrane p r e p a r a t i o n as w e l l as i n a s o l u b l e 200 000 g s u p e r n a t a n t f r a c t i o n o f e n d o t h e l i a l c e l l s . Enzyme t e s t s were p e r formed u s i n g s t a n d a r d p r o c e d u r e s , s u b s t r a t e s and p r o d u c t s were sep a r a t e d by HPLC.
RESULTS AND DISCUSSION
I n T a b l e 1 mean v a l u e s f r o m t h r e e i n d i v i d u a l s e r i e s o f a n a l y ses c o n c e r n i n g c o n t e n t s o f p u r i n e n u c l e o t i d e s , n u c l e o s i d e s and bases i n non- g r o w i n g c o n f l u e n t e n d o t h e l i a l c e l l c u l t u r e s a r e l i s t e d . For reasons o f comparison r e s p e c t i v e d a t a f o r normoxic m y o c a r d i a l t i s s u e a r e a l s o g i v e n . O b v i o u s l y , e n d o t h e l i a l c e l l s c o n t a i n e x t r a o r d i n a r i l y h i g h amounts o f ATP, ADP and AMP. The sum o f t h e adenine n u c l e o t i d e s (£?ATP, ADP, AMP) reaches w i t h more t h a n 15 umoles/g a v a l u e w h i c h i s about t h r e e t i m e s h i g h e r t h a n t h e mean ade n i n e n u c l e o t i d e c o n t e n t o f c a r d i a c t i s s u e . A n o t h e r i n t e r e s t i n g f e a t u r e o f e n d o t h e l i a l c e l l s concerns t h e i r h i g h l e v e l s o f adenine n u c l e o t i d e d e g r a d a t i v e s . The c o n t e n t s o f a d e n o s i n e , i n o s i n e , a d e n i n e and h y p o x a n t h i n e a r e about 1 t o 2 o r d e r s o f magnitude h i g h e r t h a n t h e r e s p e c t i v e v a l u e s f o r t h e myocardium. I n c o n t r a s t t o t h e h i g h l e v e l s o f adenine n u c l e o t i d e s g u a n i n e n u c l e o t i d e s a r e p r e s e n t i n end o t h e l i a l c e l l s o n l y i n s m a l l q u a n t i t i e s , w h i c h a r e s i m i l a r t o th o s e i n m y o c a r d i a l and o t h e r t i s s u e s .
PURINE M E T A B O L I S M IN C U L T U R E D E N D O T H E L I A L C E L L S 27
T a b l e 1: C o n t e n t o f ade n i n e n u c l e o t i d e s and t h e i r d e p h o s p h o r y l a t e d d e g r a d a t i v e s i n c o n f l u e n t c o r o n a r y e n d o t h e l i a l c e l l s and i n myocard i a l t i s s u e o f g u i n e a p i g s . Mean v a l u e s f r o m t h r e e i n d i v i d u a l s e r i e s of a n a l y s e s o f 11 c u l t u r e d i s h e s each.
E n d o t h e l i a l c e l l s Myocardium nmoles/g nmoles/g
ATP 11 960 4 280 ADP 2 760 1 050 AMP 630 160
^> fATP, ADP, AMP 1 5 350 5 490
Adenosine 87 2 I n o s i n e 100 1 .2 Adenine 60 0.5 Hypoxanthine 50 0.9
GTP 233 200 GDP 157 100 GMP 37 23 Guanosine *) *) Guanine *) *)
not d e t e c t a b l e
A d d i t i o n a l e x p e r i m e n t s r e v e a l e d t h a t g r o w t h s t a t e o f t h e c u l t u r e s d i d n o t p r o f o u n d l y i n f l u e n c e t h e t o t a l c o n t e n t o f adenine n u c l e o t i d e s . F u r t h e r m o r e , i n c u b a t i o n o f c o n f l u e n t c e l l c u l t u r e s i n p u r i n e - f r e e media f o r t h r e e days d i d n o t r e s u l t i n any d e t e c t a b l e r e d u c t i o n o f t h e adenine n u c l e o t i d e c o n t e n t . On t h e o t h e r hand, e n d o t h e l i a l c e l l s p r o v e d t o be s e n s i t i v e t o l a c k o f oxygen. I t i s e v i d e n t f r o m t h e d a t a i n F i g . 1 t h a t b r i e f p e r i o d s o f a n o x i c i n c u b a t i o n (1.5 and 3 m i n , r e s p e c t i v e l y ) cause a pronounced decrease o f ATP w i t h a c o r r e s p o n d i n g i n c r e a s e i n ADP and AMP l e v e l s . S i m u l t a n e o u s l y , r e m a r k a b l e amounts o f adenosine a r e formed and r e l e a s e d f r o m t h e c e l l s i n t o t h e i n c u b a t i o n medium.
I t appears f r o m a l l t h e s e o b s e r v a t i o n s t h a t t h e e x t r e m e l y h i g h a d e n i n e n u c l e o t i d e l e v e l s a r e v e r y l i k e l y a s p e c i f i c f e a t u r e o f c u l t u r e d e n d o t h e l i a l c e l l s . T h i s v i e w i s f u r t h e r s u p p o r t e d by det e r m i n a t i o n s o f a c t i v i t y v a l u e s o f enzymes i n v o l v e d i n d e g r a d a t i o n and s y n t h e s i s o f ade n i n e n u c l e o t i d e s ( T a b l e 2 ) . W h i l e 5 1 - n u c l e o t i dase a c t i v i t y i n e n d o t h e l i a l c e l l s exceeds by f a r t h a t o f myocar-
28 S. N E E S E T A L
NORMOXIA ANOXIA
10 1.5min 3min
l nmoles/g ]
F i g . 1: I n f l u e n c e o f a n o x i a on l e v e l s o f adenine n u c l e o t i d e s i n cor o n a r y e n d o t h e l i a l c e l l s and on t h e r e l e a s e o f adenosine i n t o t h e medium (mean v a l u e s f r o m 3 e x p e r i m e n t s )
d i a l t i s s u e , t h e o p p o s i t e h o l d s t r u e f o r adenosine deaminase, t h e a c t i v i t y o f w h i c h i s much h i g h e r i n t h e myocardium. These d i f f e r e n ces i n t h e p a t t e r n of enzyme a c t i v i t i e s may r e a s o n a b l y e x p l a i n t h a t e n d o t h e l i a l c e l l s c o n t a i n adenosine i n r a t h e r h i g h amounts compared w i t h t h e s m a l l q u a n t i t i e s o f t h i s n u c l e o s i d e f o u n d i n t h e myocardium. As i s f u r t h e r e v i d e n t f r o m t h e d a t a i n T a b l e 2, a c t i v i t i e s o f G-6-PDH and PRPP-synthetase, i n d i r e c t l y i n v o l v e d i n t h e b i o s y n t h e s i s o f n u c l e o t i d e s , p r o v e d t o be much h i g h e r i n e n d o t h e l i a l c e l l s t h a n i n c a r d i a c t i s s u e . These f i n d i n g s are i n accordance w i t h r e s u l t s f r o m p r e l i m i n a r y s t u d i e s , i n w h i c h by use o f 1-^C-g l y c i n e and ^ C - l a b e l e d p u r i n e bases p u r i n e n u c l e o t i d e s y n t h e s i s i n e n d o t h e l i a l c e l l s was shown t o p r o c e e d v i a s a l v a g e and de novo pathways.
SUMMARY
E n d o t h e l i a l c e l l s f r o m c o r o n a r y v e s s e l s o f g u i n e a p i g h e a r t s were i s o l a t e d , c u l t i v a t e d and m o r p h o l o g i c a l l y c h a r a c t e r i z e d . - C e l l s
P U R I N E M E T A B O L I S M IN C U L T U R E D E N D O T H E L I A L C E L L S 29
T a b l e 2: Enzyme a c t i v i t i e s i n c u l t u r e d c o r o n a r y e n d o t h e l i a l c e l l s and i n c a r d i a c t i s s u e f r o m g u i n e a p i g s .
S p e c i f i c a c t i v i t y [ n m o l e s / m i n *mg]
E n d o t h e l i a l c e l l s
C a r d i a c t i s s u e
5 ' - N u c l e o t i d a s e [E.C. 3.1.3.5] 95 13.6
A l k a l i n e p h o s p h a t a s e [E.C. 3.1.3.1] 14.6 24
AMP d e a m i n a s e [E.C.3.5.4.6] 1 .1 2.2
A d e n o s i n e d e a m i n a s e [E.C. 3.5.4.4] 3.4 28
G l u - 6 - P - d e h y d r o g e n a s e [E.C. 1.1.1.49] 12.7 4.2
P R P P - s y n t h e t a s e [E.C. 2.7.6.6] 6.58 1 .9
A P R - t r a n s f e r a s e [E.C. 2.4.2.7] 0.7 0.2
G P R - t r a n s f e r a s e [E.C. 2.4.2.8] 0.3 0.1
A d e n y l a t e c y c l a s e [E.C. 4.6.1.1] 0.1 0.4
P h o s p h o d i e s t e r a s e [E.C. 3.1.4.1] 2.1 15
f r o m c o n f l u e n t c u l t u r e s c o n t a i n e d adenine n u c l e o t i d e s and t h e i r de-p h o s p h o r y l a t e d d e g r a d a t i v e s i n e x c e p t i o n a l l y h i g h amounts.- Adenine n u c l e o t i d e l e v e l s were o n l y s l i g h t l y i n f l u e n c e d by t h e g r o w t h s t a t e o f t h e c u l t u r e s and remained s t a b l e d u r i n g i n c u b a t i o n f o r t h r e e days i n p u r i n e - f r e e medium. I n c o n t r a s t , b r i e f i n c u b a t i o n o f endot h e l i a l c e l l s under a n o x i c c o n d i t i o n s r e s u l t e d i n a s u b s t a n t i a l breakdown o f adenine n u c l e o t i d e s a s s o c i a t e d w i t h an enhanced f o r m a t i o n and r e l e a s e o f a d e n o s i n e . - Measurements o f s p e c i f i c a c t i v i -
30 S. N E E S E T A L
t i e s o f enzymes i n v o l v e d i n adenine n u c l e o t i d e s y n t h e s i s and deg r a d a t i o n l e n d a d d i t i o n a l s u p p o r t t o t h e v i e w t h a t a v e r y a c t i v e a d e n i n e n u c l e o t i d e m e t a b o l i s m i s a t y p i c a l f e a t u r e o f c u l t u r e d c o r o n a r y e n d o t h e l i a l c e l l s .
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